Energy compensated spectrofluorometer

ABSTRACT

An energy-compensated spectrofluorometer of the time-shared double beam type employing a rotating chopper mirror disc providing a main measure channel as one of the time-shared outputs of a photomultiplier tube and a compensating reference channel generated by a reference light whose output is conveyed by a light pipe to the cathode of the photomultiplier tube, generating the other time-shared output thereof. The energy correction is derived from a thermal detector cell receiving part of the excitation beam, and the reciprocal of the thermal detector cell signal is subtracted from the dark currentcorrected reference channel signal and is employed to adjust the high voltage supplied to the photomultiplier tube, thereby adjusting its gain. The measure channel includes means to compensate the measure signal for non-uniform spectral sensitivity of the photomultiplier tube, as well as for dark current. The reference channel signal is also modified to compensate for wavelength-dependent variations of quantum intensity in the excitation beam.

United States Patent Kremen et al.

l l ENERGY COMPENSATED SPECTROFLUOROMETER [75] Inventors: Jerome C.Kremen, Takoma Park;

Isaac Landa, Wheaton. both of Md.

{73] Assignee: Baxter Laboratories. Inc., Morton Grove. Ill.

[22] Filed: Mar. 20, 1974 [2!] Appl. No.: 452,980

[52] US. Cl 250/458; 250/46l [51] Int. Cl. ..G01n 21/52 [58] Field ofSearch 250/301. 304. 363, 372, 250/393, 458. 461; 356/96 l 56]References Cited UNITED STATES PATENTS 3.465.l43 9/l969 Doonan 250/372 X3.497.690 2/l970 Wheeless. Jr. ct al 250/304 3.795.918 3/[974 Sunderlandi. 356/96 X 3.832.555 8/l974 Ohnishi 250/458 Primary E.\'an1inerArchieR. Borchelt Attorney. Agent, or FirmHerman L. Gordon;

Richard G. Kinney [lll 3,891,853

[ June 24, 1975 [57] ABSTRACT An energy-compensated spectrofluorometerof the time-shared double beam type employing a rotating chopper mirrordisc providing a main measure channel as one of the time-shared outputsof a photomultiplier tube and a compensating reference channel generatedby a reference light whose output is conveyed by a light pipe to thecathode of the photomultiplier tube, generating the other timesharedoutput thereof. The energy correction is derived from a thermal detectorcell receiving part of the excitation beam, and the reciprocal of thethermal detector cell signal is subtracted from the darkcurrent-corrected reference channel signal and is employed to adjust thehigh voltage supplied to the photomultiplier tube, thereby adjusting itsgain. The measure channel includes means to compensate the measuresignal for non-uniform spectral sensitivity of the photomultiplier tube.as well as for dark current. The reference channel signal is alsomodified to compensate for wavelength dependent variations of quantumintensity in the exci tation beam.

14 Claims, 4 Drawing Figures SHEET PATENTEDJUN 24 ms 85038 9 A/CE a2RAM/3M I r rAA/(E ENERGY COMPENSATED SPECTROFLUOROMETER This inventionrelates to spectrophotometers, and more particularly to anenergy-compensated spectrofluorometer of the time-shared double beamtype.

A main object of the invention is to provide a novel and improvedspectrophotometer which may be employed in various different modes,including spectrofluorometer modes, spectral absorbance modes,differential spectral fluorometry modes, and others, and which iscompensated for differences in energy from its exci tation source atdifferent wavelengths, and which is also compensated for differences inwavelength sensitivity of its photo-sensing means, the spectrophotometerbeing simple in construction, being stable in operation, and includingmeans to compensate for the absorbance effect of the liquid contained inits sample holder.

A further object of the invention is to provide an improvedenergy-compensated spectrofluorometer of the time-shared double-beamtype which may be employed in various modes, the spectrofluorometerhaving means to scan a sample with light of variable wavelength andhaving means to provide the effect of equal excitation energy at eachwavelength, and also being provided with means to compensate thephotomultiplier tube thereof for differences in wavelength sensitivitythereof, the apparatus being relatively compact in size, being easy tooperate, and permitting rapid and accurate assays of the opticalproperties of materials.

A still further object of the invention is to provide an improvedspectrofluorometer device of the time-shared double beam type which isprovided with means for successively exciting a sample with all thespectral components of a polychromatic light source for determining thecharacteristic wavelength or wavelengths at which the sample has majorfluorescent response and for quantitatively analyzing such majorresponse for each of such excitation wavelengths, the double beamspectrofluorometer including highly effective means for generating areference channel from the same photomultiplier tube employed togenerate its measure channel, including means for automaticallycomputing and applying corrections for different excitation energies atthe different excitation wavelengths and being provided with means forapplying a further correction factor to take care of variations inemission response sensitivity of the apparatus at different emissionwavelengths, and being further provided with means for applyingcorrections to compensate for variations in quantum intensity withwavelength for the excitation beam spectrum, and for variations in thequantum emission of the sample as a function of emission wavelength, sothat a complete and accurate quantitative assay of the fluorescentproperties of a sample may be obtained, and so that the material may beaccurately identified, the apparatus requiring the use of only a smallsample of the material to be analyzed, being highly sensitive, beingapplicable for identifying and quantitatively analyzing a wide range offluorescent materials, and permitting the continuous activation of asample and the measurement of resulting fluorescence throughout a wideradiant energy spectrum.

Further objects and advantages of the invention will become apparentfrom the following description and claims, and from the accompanyingdrawings, wherein:

FIG. 1 is a schematic diagram of an improved timesharingenergy-compensated double beam spectrofluorometer constructed inaccordance with the present invention.

FIG. 2 is a horizontal plan view of the rotating chopper mirror discemployed in the spectrofluorometer of HO. 1, said view being takensubstantially on the line 2-2 of FIG. 1.

FIG. 3 is a diagram showing how the device of FIG. 1 may be modified foruse as a spectrophotometer for making absorbance or transmittancemeasurements.

FIG. 4 is a diagram showing how the device of FIG. 1 may be modified foruse as a differential spectrofluorometer.

Referring to the drawings, 10 generally designates a spectrofluorometerof the double beam type employing a photomultiplier tube 1 l as itsphotosensitive element, said tube having a cathode 12, an anode l3 andother well known elements, not shown. The photomultiplier tube 11 issupplied with high voltage from an adjustable high voltage supply source14 whose output 15 is shown as typically connected to cathode 12. Thenegative output voltage at 15 can be controlled in a conventional mannerin accordance with an input control signal at 16. The gain of thephotomultiplier tube II will be controlled in accordance with thevoltage delivered to cathode 12.

The spectrofluorometer 10 includes a transparent sample cell 17 arrangedto receive an excitation beam 18 from a first variable monochromator 19which receives input radiation from a suitable wide-range source 20. Themonochromator l9 emits a beam 21 of selected excitation wavelength (orvarious wavelengths in sequence when excitation scanning or excitationsequentially with multiple excitation wavelengths is desired), and atimed portion of beam 21 is reflected as beam 18 from a rotating choppermirror disc 22, presently to be described, and is thus directed towardthe sample cell 17. The disc 22 allows a second timed portion 23 of theexcitation beam to reach a stationary plane mirror 24 positioned so asto reflect beam portion 23 through a compensating cell 25 to a thermaldetector 26 which will generate a signal voltage E whose strength is inaccordance with the energy level of the received beam. This device 26may be a surface charge device wherein the charge difference across thedevice varies with temperature changes, similar to Model No. [CT-2030,manufactured by Laser Precision Corp., Yorkville, NY. This device has aresponse to energy substantially independent of wavelength, i.e.,provides a substantially flat spectral response.

In the typical spectrofluorometer arrangement illustrated in FIG. 1, afluorescence emission beam 27 from the fluorescent sample in cell 17 isdirected onto the rotating chopper mirror disc 22 and is reflectedtherefrom, in the same time segment as beam 18, to a stationary curvedtoroidal mirror 28, from which it is reflected into a second variablemonochromator 29. A selected wavelength emission beam 30 frommonochromator 29 is directed to the cathode 12 of the photomultipliertube 11. The response signal from the photomultiplier tube will then bein accordance with the strength of the selected wavelength emission beam30 (E and with wavelength-dependent factors characteristic of theoptical path and the photomultiplier tube, respectively, (T and PM A Areference channel synchronized with beam 23 is derived from a referencelight source 31, which may comprise a source similar to Model No. MLEDSSmanufactured by Motorola Semiconductor Products, Inc., Phoenix, Ariz.,which provides substantially red emission (about 660 nm). This type ofreference source is preferred since the photomultiplier tube is nothighly sensitive to red and therefore this avoids scatter lightinterference. The output of source 31 is directed through a first lightpipe segment 32 through the chopper disc 22 to an end 34 of a secondlight pipe segment 33 aligned with the first segment 32. The secondlight pipe segment 33 leads to the photomultiplier tube 11 and has aninclined exit end portion 35 adjacent to and directed toward cathode 12.The reference light from source 31 thus provides an output signal fromtube 11 synchronized with the thermal detector signal E previouslymentioned.

Synchronized switching means, designated generally at 36, is employed toalternately switch the output of the photomultiplier tube to a measurechannel, shown at 37, and to a reference channel, shown at 38. Theswitching means may be either electronic or mechanical, and isillustrated diagrammatically as being driven by the chopper drive motor39 for simplicity. it will be understood that a wide range of equivalentcoupling synchronizing means between the switch device 36 and therotating chopper disc 22 may be employed.

Referring to FIG. 2, it will be seen that the chopper disc 22 hasdiametrically opposed notches 40,40 which simultaneously provideexposure of thermal detector 26 to beam 21 and light pipe segment 33 toreference light 31. Between the notches 40,40 there are respective pairsof dark light-absorbing spaces 41,41 located on opposite sides ofreflecting mirror spaces 42. Mirror spaces 42,42 simultaneously providethe reflected excitation beam 18 and the reflection of emission beam 27toward the fixed curved directing mirror 28.

The compensating cell 25 (or A path cell) located ahead of thermaldetector 26 compensates for the attenuation of beam 18 in sample cell 17caused by the sample liquid. The beam 18 is attenuated by an amountproportional to one haif the path length in cell 17. Thus, the thermaldetector signal is attenuated to the same degree by using a transparentA path cell 25 containing liquid providing the same absorbancy asonehaif the path length in cell 17. For example, for a sample cell 17 ofi mm thickness, a transparent compensating cell 25 of mm thicknesscontaining the same liquid or material of the same absorbancy as thesample may be employed.

The measure output signal in channel 37 is processed through acurrent-to-voltage amplifier 43 and a base line correction amplifier 44,which is coupled to the wavelength control element of the emissionmonochromator 29 in a known manner and which is programmed to provide awavelength correction term corresponding to T h PM A and which dividesthe input signal entering device 44 by this term. A corresponding baseline correction device is shown in US. Pat, No. 3,433,952 to H. K.Howerton. Thus, device 44 makes the necessary corrections to compensatefor the abovementioned emission wavelength-dependent factors. The device44 may be accompanied by a unit conversion device 45 which introduces again change proportional to wavelength or wavelength cubed to convertthe corrected signal to desired units, such as number of quanta pernanometer of bandwidth, number of quanta per CM (wave number) ofbandwidth, or the like.

After the above-described processing, the resultant measure signalpasses through a dark current correction device 46, which subtracts theresidual photomui tipiier dark current (the component E,, of the measuresignal produced by dark space 41) from the input measure signal, and thedark current'corrected signal then passes through an output amplifier 47to the instrument display devices, such as a recorder and meter.

The reference light output signal in channel 38 is pro cessed through acurrent-to-voitage amplifier 48 and a quantum intensity correctiondevice 49 which is cou' pied to the wavelength control element of theexcitation monochromator 19 and which is programmed to apply acorrection term to compensate for wavelengthdependent quantum intensityvariations in the excitation beam 21 by introducing a gain changeproportional to the excitation wavelength.

The quantum-corrected signal is then corrected for dark current in adark current subtractor 50 similar to subtractor 46 (dark current signalcomponent E is subtracted), and the resultant dark current-correctedreference light signal E is furnished to one input of a subtractor 51.

The thermal detector signal E is processed through an inverter device 52providing its reciprocal i/E at its output. Said reciprocal signal isfurnished to the other input of the subtractor 51, and the resultantsubtractor output comprising the energy-compensated remainder signal Ei/E is delivered through a suitable limiter 53 to the high voltagesupply control input 16.

The above-processed reference light signal is thus employed to vary thehigh voltage furnished by the supply unit 14 and thereby vary the gainof the photomultiplier tube 11 in a manner to compensate for i energyvariations in the excitation beam 21, and (2) wavedependent quantumintensity variations in the excitation beam.

FIG. 3 diagrammatically illustrates how the spectrofluorometcr of FIG. 1may be modified for use as an instrument for making absorbance ortransmittance measurements. in this arrangement, the excitation" beam 18travels through the sample cell 17 and is then reflected back to thechopper mirror disc from a stationary plane mirror 60 located parallelto rotating chopper disc 22, defining a reflected beam 27', taking apath substantially similar to that taken by the emission beam 27 in thepreviously described embodiment. The emission" monochromator 29 eithermay be omitted or may be set at the wavelength of the excitation beam21. in the arrangement shown in FiG. 3, spectral absorbance ortransmittance measurements may be made through a sample in cell 17.

FIG. 4 diagrammatically illustrates how the spectrofluorometer may bemodified for use as a differential double beam spectrofluorometer. Afirst fluorescent sample in cell 17 is excited to fluorescence by achopper-timed excitation beam reflected from the subjacent mirrorelement 42 of the chopper mirror disc 22 and an emission beam 27 issimilarly reflected from the opposite mirror element 42 of the choppermirror disc in the same manner as in FIG. 1, passing through themonochromator 29 and generating a response from the photomuitiplier tubedefining a first measure channel. A

timed following portion of excitation beam Zlirnmdiately thereaftertravels through a notch 40 of the disc and is reflected from a firstfixed plane mirror 6!, arranged parallel to disc 22, back through thenotch and is directed to a second transparent sample cell 62 containinga second fluorescent sample. Emission radiation is generated in cell 62and leaves as an emission beam 63, the cell 62 being suitably orientedso that beam 63 can be directed through the opposite notch 40 of disc 22to a fixed plane mirror 64 arranged parallel to disc 22 so as to reflectemission beam 63 back through said last-named notch 40 to travel alongthe same path as the reflected emission beam 27, namely, toward thefixed mirror 28, eventually to be directed through the monochromator 29to the cathode 12 of the photomultiplier tube 11. The emission beamsfrom the two samples are thus time-shared as they reach thephotomultiplier tube and provide corresponding time-shared signals whichcan be compared or measured differentially. in the arrangement of FIG.4, the reference light compensation channel, previously described inconnection with FIG. 1, is not employed.

While certain specific embodiments of improved spectrophotometers havebeen described in the foregoing description, it will be understood thatvarious modifications within the spirit of the invention may occur tothose skilled in the art. Therefore it is intended that no limitationsbe placed on the invention except as defined by the scope of theappended claims.

What is claimed is:

1. In a spectrofluorometer, a source of radiant energy, a transparentsample holder, a photosensitive detector having variable gain, a movingchopper element, means including said moving chopper element defining anexcitation optical channel directed to said holder and a second opticalchannel, means defining an emission optical channel between said sampleholder and said photosensitive detector, a measure signal channel and areference signal channel, switch means synchronized with the chopperelement to alternately connect the output of the photosensitive detectorto the measure signal channel and the reference signal channel, areference radiant energy source, means including said moving chopperelement defining an optical path between said reference source and saidphotosensitive detector time-shared with said emission optical channel,and means controlling the gain of said photosensitive detector inaccordance with the signal generated in said photosensitive detector bysaid reference radiant energy source and the energy content of saidsecond optical channel.

2. The spectrofluorometer of claim 1, and wherein said first-namedsource includes variable excitation wavelength-selection means, andmeans to apply a quantum intensity correction to the reference signalchannel in accordance with the wavelength setting of said variableexcitation wavelength-selection means.

3. The spectrofluorometer of claim 1, and wherein said emission opticalchannel includes variable emission wavelength-selection means, and meansto apply an emission wavelength-dependent correction to said measuresignal channel in accordance with the wavelength setting of saidvariable emission wavelengthselection means.

4. In a spectrofluorometer, a source of radiant energy, a transparentsample holder, a thermal electric detector means, means including amoving chopper element defining alternate time-spaced optical paths re-'spectively between said source and said sample holder and between saidsource and said thermal electric detector means, whereby to generate anenergy correction signal, a photosensitive detector, means defining athird optical path between said sample holder and said photosensitivedetector, whereby to generate a measure signal at the output of saidphotosensitive detector, a reference radiant energy source, meansincluding said chopper element defining a fourth optical path betweensaid last-named source and said photosensitive detector which istime-spaced relative to said third optical path, whereby to generate areference signal at the output of the photosensitive detector which istimespaced relative to said measure signal, and means to control theresponse of said photosensitive detector in accordance with both saidreference signal and said energy correction signal.

5. The spectrofluorometer of claim 4, and wherein the means to controlthe response of said photosensitive detector comprises computing meansto derive the reciprocal of said energy signal and to compare saidreference signal with said reciprocal.

6. The spectrofluorometer of claim 5, and wherein said photosensitivedetector comprises a photomultiplier having an adjustable high voltagesupply source, and wherein the means to control the response of thephotosensitive detector comprises means to adjust said high voltagesupply source in accordance with the difference between said referencesignal and said reciproca].

7. The spectrofluorometer of claim 6, and an output channel providedwith indicator means, and a reference channel, and means synchronizedwith said moving chopper element to alternately connect the output ofthe photomultiplier to said output channel and said reference channel,said reference channel including said computing means and beingoperatively connected to said adjustable high voltage supply source.

8. The spectrofluorometer of claim 6, and wherein said means definingsaid third optical path includes said moving chopper element.

9. The spectrofluorometer of claim 8, and wherein said means definingthe fourth optical path includes a light pipe having an inlet endreceiving the output of said reference radiant energy source and havingan outlet end adjacent to and operatively directed toward saidphotomultiplier.

10. The spectrofluorometer of claim 4, and wherein said first-namedradiant energy source includes variable excitation wavelength-selectionmeans, and means to apply a quantum intensity correction to saidreference signal in accordance with the wavelength setting of saidvariable wavelength-selection means.

11. The spectrofluorometer of claim 4, and wherein said means definingthe third optical path includes variable emission wavelength selectionmeans, and means to apply an emission wavelength-dependent correction tosaid measure signal in accordance with the setting of said variableemission wavelength-selection means.

12. The spectrofluorometer of claim 4, and an output channel providedwith indicating means, and a reference channel, said photosensitivedetector comprising a photomultiplier having an adjustable high voltagesupply means, switching means synchronized with said moving chopperelement to alternately connect the output of said photomultiplier tosaid output channel and said reference channel, the means to control theresponse of said photosensitive detector comprising computing means toderive the reciprocal of said energy signal, and computing means in saidreference channel to derive a comparison signal comprising thedifference between said reference signal and said reciprocal, andcircuit means to operatively connect said comparison signal to saidadjustable high voltage supply means.

13. The spectrofluorometer of claim 12, and wherein said first-namedradiant energy source includes variable excitation wavelength-selectionmeans, means to apply a quantum intensity correction to said referencesignal in said reference channel ahead of said secondnamed computingmeans, and wherein said means defining the third optical path includesvariable emission wavelength-selection means, and means to apply anemission wavelength-dependent correction to said measure signal in saidoutput channel in accordance with the setting of said variable emissionwavelengthselection means.

14. The spectrofluorometer of claim 4, and a transparent absorbancycompensation cell in said optical path between said first-named sourceand said thermal electric detector means, said cell having substantiallyone-half the thickness of the sample holder and being adapted to containmaterial substantially of the same absorbancy as that inserted in thesample holder.

1. In a spectrofluorometer, a source of radiant energy, a transparentsample holder, a photosensitive detector having variable gain, a movingchopper element, means including said moving chopper element defining anexcitation optical channel directed to said holder and a second opticalchannel, means defining an emission optical channel between said sampleholder and said photosensitive detector, a measure signal channel and areference signal channel, switch means synchronized with the chopperelement to alternately connect the output of the photosensitive detectorto the measure signal channel and the reference signal channel, areference radiant energy source, means including said moving chopperelement defining an optical path between said reference source and saidphotosensitive detector time-shared with said emission optical channel,and means controlling the gain of said photosensitive detector inaccordance with the signal generated in said photosensitive detector bysaid reference radiant energy source and the energy content of saidsecond optical channel.
 2. The spectrofluorometer of claim 1, andwherein said first-named source includes variable excitationwavelength-selection means, and means to apply a quantum intensitycorrection to the reference signal channel in accordance with thewavelength setting of said variable excitation wavelength-selectionmeans.
 3. The spectrofluorometer of claim 1, and wherein said emissionoptical channel includes variable emission wavelength-selection means,and means to apply an emission wavelength-dependent correction to saidmeasure signal channel in accordance with the wavelength setting of saidvariable emission wavelength-selection means.
 4. In aspectrofluorometer, a source of radiant energy, a transparent sampleholder, a thermal electric detector means, means including a movingchopper element defining alternate time-spaced optical pathsrespectively between said source and said sample holder and between saidsource and said thermal electric detector means, whereby to generate anenergy correction signal, a photosensitive detector, means defining athird optical path between said sample holder and said photosensitivedetector, whereby to generate a measure signal at the output of saidphotosensitive detector, a reference radiant energy source, meansincluding said chopper element defining a fourth optical path betweensaid last-named source and said photosensitive detector which istime-spaced relative to said third optical path, whereby to generate areference signal at the output of the photosensitive detector which istime-spaced relative to said measure signal, and means to control theresponse of said photosensitive detector in accordance with both saidreference signal and said energy correction signal.
 5. Thespectrofluorometer of claim 4, and wherein the means to control theresponse of said photosensitive detector comprises computing means toderive the reciprocal of said energy signal and to compare saidreference signal with said reciprocal.
 6. The spectrofluorometer ofclaim 5, and wherein said photosensitive detector comprises aphotomultiplier having an adjustable high voltage supply source, andwherein the means to control the response of the photosensitive detectorcomprises means to adjust said high voltage supply source in accordancewith the difference between said reference signal and said reciprocal.7. The spectrofluorometer of claim 6, and an output channel providedwith indicator means, and a reference channel, and means synchronizedwith said moving chopper element to alternately connect the output ofthe photomultiplier to said output channel and said reference channel,said reference channel including said computing means and beingoperatively connected to said adjustable high voltage supply source. 8.The spectrofluorometer of claim 6, and wherein said means defining saidthird optical path includes said moving chopper element.
 9. Thespectrofluorometer of claim 8, and wherein said means defining thefourth optical path includes a light pipe having an inlet end receivingthe output of said reference radiant energy source and having an outletend adjacent to and operatively directed toward said photomultiplier.10. The spectrofluorometer of claim 4, and wherein said first-namedradiant energy source includes variable excitation wavelength-selectionmeans, and means to apply a quantum intensity correction to saidreference signal in accordance with the wavelength setting of saidvariable wavelength-selection means.
 11. The spectrofluorometer of claim4, and wherein said means defining the third optical path includesvariable emission wavelength selection means, and means to apply anemission wavelength-dependent correction to said measure signal inaccordance with the setting of said variable emissionwavelength-selection means.
 12. The spectrofluorometer of claim 4, andan output channel provided with indicating means, and a referencechannel, said photosensitive detector comprising a photomultiplierhaving an adjustable high voltage supply means, switching meanssynchronized with said moving chopper element to alternately connect theoutput of said photomultiplier to said output channel and said referencechannel, the means to control the response of said photosensitivedetector comprising computing means to derive the reciprocal of saidenergy signal, and computing means in said reference channel to derive acomparison signal comprising the difference between said referencesignal and said reciprocal, and circuit means to operatively connectsaid comparison signal to said adjustable high voltage supply means. 13.The spectrofluorometer of claim 12, and wherein said first-named radiantenergy source includes variable excitation wavelength-selection means,means to apply a quantum intensity correction to said reference signalin said reference channel ahead of said second-named computing means,and wherein said means defining the third optical path includes variableemission wavelength-selection means, and means to apply an emissionwavelength-dependent correction to said measure signal in said outputchannel in accordance with the setting of said variable emissionwavelength-selection means.
 14. The spectrofluorometer of claim 4, and atransparent absorbancy compensation cell in said optical path betweensaid first-named source and said thermal electric detector means, saidcell having substantially one-half the thickness of the sample holderand being adapted to contain material substantially of the sameabsorbancy as that inserted in the sample holder.